Sioorgmic & Medicinal ChemistryL.etters. Vol.2. No.1. pp. 91-94, 1992 Prited in Great Britain

0960-894X/92 S3.M) + .oO @ 1992 Pergamon Press pk

Synthesis of a Photoreactive Taxol Side Chain

Arindam Chatterjee,a John S. Williamson,a Jordan K. Zjawiony,h John R. Petersonh*

a Department of Medicinal Chemisuy, h Department of Pharmacognosy and the Research Institute of

Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677,

USA

(Received 25 September 1991)

Abstract: The photoreactive taxol side chain, @R,3S)-(-)-N-(4’-azido-benzoyl-3-phenylisoserine methyl ester (6), was prepared in four steps from (2R,3R)-(+)-methyl3-phenylglycidate (2).

Tax01 (l), a potent antimitotic natural product isolated from the bark of the western yew, Taxus

brevifolia, has shown promising clinical activity against ovarian cancer. l-3 Although the unique abilities of taxol

to promote the assembly of tubulin to microtubules and to stabilize the intact microtubule assembly against

depolymerization have been well documented in vitro, very little is known about the location and molecular

characteristics of the protein binding site(s) for taxo1.3-5

1 (Taxol)

Photoaffiiity labelling is an excellent tool for the identification and characterization of ligand-specific

mediators of many biological and pharmacological phenomena. 6.7 Significant progress has been made over the

past decade in understanding the interaction of biologically active compounds with neuroreceptors, hormone

receptors, transport proteins, nucleic acids and nucleotides, and enzymes through use of the photoaffinity labelling

technique. Surprisingly little has been done with tubulin or tubulin/microtubule binding agents, however. Two

well known antimitotic agents, colchicine and vinblastine, have been converted to photoreactive derivatives and

employed in photoaffmity labelling studies of tubulin. g-11 Kingston, et al. reported recently a photoreactive

* Present address: Panlabs Inc., Bothell, WA 9801 l-8805

91

92 A. CHA-ITERJEE et al.

derivative of taxol that incorporated a C-ring 7-azibenzoyl group. 3 The aromatic rings of the taxol side chain are

potentially two additional sites for photoreative functional group incorporation, particularly since this modification

would be expected to have a minimal affect upon the biological activity of the taxol or taxane analog.1-35***-14

Herein we wish to report the synthesis of a photoreactive taxol side chain that bears an azido group at the 4’-

position of the N-benzoyl ring.

Our synthesis of the photoreactive taxol side chain, (2R,35’)-(-)-N-(4’-azidobenzoyl)-3-phenylisosexine

methyl ester (6), is outlined in Scheme 1. Subjection of the (+)-glycidic ester 2 to the sodium azide ring opening

methodology of Denis et al. delivered the (2&3S)-azido alcohol 3 in 93% yield on an 0.15 mol scale.15J6 The

reported tedious chromatographic purification of 3 could be avoided, however, through a modified workup

procedure in which the reaction solvent mixture was concentrated and the product extracted into ether. The azido

alcohol 3 obtained upon evaporation of the extract was sufficiently pure (>95% by ‘H NMR) for subsequent use.

Incorporation of an azidobenzamide group into the taxol side chain relied upon the known propensity for 0 to N

aroyl group transfer.15 In this regard, 3 was converted first to the 4’-nitrobenzoate 4 by treatment with 4-

nitrobenzoyl chloride and triethylamine in the presence of a catalytic amount of N,N-dimethylaminopyridine.

Catalytic reduction of 4 over 10% palladium on carbon produced 0-(4’-aminobenzoyl)-3-phenylisoserine methyl

ester, which rearranged spontaneously over the course of 2 days to the I’aminobenzamide 5. Compound 5 was

Ph 4’-NO,C,H,COCl,

C!OzMe EtaN, DMAP, cH,Clz

N3 / i l-2 h, 88%

50 “C, 46 h, 93% 6H

3

Ph Ph

I\, C&Me

Ha, 10% Pd/C -* 4’-NH&H&ONH L

COzMe

I’73 : i MeOH, 72 h, 98% i

4’-N02C6H&00 OH

4 5

Ph

NaNOa, HOAc; NaNa 4’-N,CeH,CONH L

CqMe *

O-10 ‘C, 2-3 h, 70% I

XH 6

Scheme 1

A photoreactive tax01 side chain 93

only slightly soluble in methanol and consequently the product crystallized directly from the reaction mixture.

Introduction of the photolabile azido group was accomplished in 70% yield by mild oxidation of 5 with 1.1 mol

equivalents of sodium nitrite in an acetic acid solution of sodium azide. The 4’-azido taxol side chain 6 exhibited

strong absorptions in the IR spectrum at 2120 cm-* and in the UV spectrum (EtOH) at 205 nm (log E 4.43) and

269 nm (log E 4.32).

In summary, the photoreactive taxol side chain 6 was prepared from (2R,3R)-(+)-methyl 3-

phenylglycidate in 56% overall yield and in a form suitable for coupling to the baccatin III nucleus of taxol.l**lg

Furthermore, the 4’-aminobenzamide side chain 5 permits the introduction of a radiolabel into the molecule or the

synthesis of other photoreactive analogs by condensation with commercial photoreactive derivatizing agent&s7

such as N-hydroxysuccinimidyl-4-azidobenzoate and 6-(4-azido-2-nitrophenylamino)hexanoic acid N-

hydroxysuccin-imide. Our continued efforts with the amino and azido side chains will be the subject of future

reports.

Acknowledgement. The generous financial support of the Elsa U. Pardee Foundation is graciously

acknowledged. AC and JSW also thank Dr. Manuel G. Sierra for his helpful discussions.

References and Notes

(1) Suffness, M. Gann Monogr. Cancer Res. 1989,36,21&I.

(2) Suffness, M.; Cordell, G.A. In The Alkaloids, Chemistry andPharmacology; Brossi, A., Ed.; Academic

Press: Orlando, Florida, 1985; Vol. Xxv, pp l-18 and 280-288.

(3) Kingston, D.G.I.; Samaranayake, G.; Ievy, C.A. J. Nat. Prod. 1990.53, 1.

(4) Rowinsky, E.K.; Cazenave, R.C.; Donehower, R.C. J. Natl. Cancer Inst. 1990,82. 1247.

(5) Pamess, J.; Kingston, D.G.I.; Powell, R.G.; Harracksingh, C.; Horwitz, S.B. Biochem. Biophys. Res .

Commun.1982,105, 1082.

(6) Schuster, D.J.; Probst, W.C.; Ehrlich, G.K.; Singh, G. Photo&em. Photobiol. 1989,49,785.

(7) Bayley, H.; Staros, J.V. In Asides and Nitrenes, Reactivity and Utility, Striven, E.F.V., Ed.; Academic

Press: Orlando, Florida, 1984; pp 433-490.

(8) Williams, R.F.; Mumford, C.L.; Williams, G.A.; Floyd, L.J.; Aivaliotis, M.J.; Mattinez, R.A.; Robinson,

AK; Barnes, L.D. J. Biol. Chem. 1985,260, 13794.

(9) Floyd, L.J.; Barnes, L.D.; Williams, R.F. Biochem. 1989,28, 8515.

(10) Safa, A.R.; Hamel, E.; Felsted, R.L. Biochem. 1987.26,97.

(11) Safa, A.R.; Felsted, R.L. J. Biol. Chem. 1987,262. 1261.

(12) Lataste, H.; Senilh, V; Wright, M.; Guenard, D.; Potier, P. Proc. Natl. Acad. Sci. USA 1984,84,4090.

94 A. CHATERJEE er al.

(13) Swindell, C.S.; Krauss, N.E.; Horwitz, S.B.; Ringel, I. J. Med. Chem. 1991,34, 1176,

(14) Gdritte-Voegelein, F.; Guenard, D.; Lavelle, F.; Le Goff, M.-T.; Mangatal, L; Potier, P. J. Med. Chem.

1991,34, 992.

(15) Denis, J.-N.; Greene, A.E.; Serra, A.A.; Luche, M.-J. J. Org. Chem. 1986,.51,46.

(16) Denis, J.-N.; Correa, A.; Greene, A.E. J. Org. Chem. 1990,55, 1957.

(17) All new compounds were fully characterized by IR, *H and 13C NMR spectroscopy, optical rotation, and

combustion analysis. Spectroscopic data for compounds S and 6 follow where the numbers in

parentheses for the carbon resonances refer to the number of attached protons on that carbon atom as

determined from the APT and DEPT - GL experiments.

(2R,3S)-(-)-N-(4’-Aminobenzoyl)-3-phenylisoserine methyl ester

(5): mp 20.5-207 ‘C (MeOH-CHC13); IR (KBr) 3480,3380,3340,1735,1630,1610,1535,1510,1440,

1355,1320,1295,1195,1087,773,708 cm- 1; 1H NMR (MeOH-d4, 300 MHz) 6 7.62 (ddd, 1 H, J = 8.8,

4.6, 2.3 Hz), 7.43-7.24 (m, 5 II), 6.68 (ddd, 1 H, J = 8.8, 4.6, 2.3 Hz), 5.56 (d, 1 H, J = 3.5 Hz), 4.87

(br s, 4 H), 4.59 (d, 1 H, J = 3.5 Hz), 3.70 (s, 3 I-I); 13C NMR (MeOH-d4,75 MHz) 174.4 (O), 170.0 (0),

153.5 (O), 140.7 (0), 130.1 (l), 129.4 (l), 128.5 (l), 128.1 (I), 122.8 (l), 114.7 (l), 75.0 (l), 57.4 (I),

52.8 (3) [a]~o = -73.5 (c = 0.20, acetone). ppm;

(2R,3S)-(-)-N-(4’-Azidobenzoyl)-3-phenylisoserine methyl ester

(6): mp 163 ‘C dec. (CHC13-petroleum ether); IR (KBr) 3500,3370,2120, 1730, 1640, 1607, 1525, 1495,

1440,1285,1258,1190,1103,850,770,710,705 cm- 1; 1H NMR (CDCl,, 300 MHz) 6 7.77 (ddd, 1 H, J

= 8.6, 4.3, 2.2 Hz), 7.47-7.29 (m, 5 H), 7.06 (ddd, 1 H, J = 8.6, 4.3, 2.2 Hz), 6.94 (br d, 1 H, J = 8.8

Hz), 5.72 (dd, 1 H, J = 8.8, 2.1 Hz), 4.63 (d, 1 H, J = 2.1 Hz), 3.85 (s, 3 H), 3.3 (br s, 1 H); 1% NMR

(CDCl3, 75 MHz) 173.4 (0), 169.8 (0), 165.8 (0), 143.7 (0), 138.6 (0), 130.4 (O), 128.9 (l), 128.8 (1)

128.0 (l), 126.9 (l), 119.1 (l), 73.2 (l), 54.9 (l), 53.3 (3) ; [a]: = -51.1 (c = 0.09, CHQ3) ppm

Anal. Calcd for C17Hl604N4: C, 59.99; H, 4.74; N, 16.46. Found: C, 60.04; H, 5.10; N, 16.14.

(18) Denis, J.-N.; Greene, A.E.; Guenard, D.; Gukritte-Voegelein, F.; Mangatal, L; Potier, P, J. Am. Chem.

sot. 1988,110, 5917.

(19) Holton, R.A. U.S. Patent 5 015 744, 1991; Chem. Abstr. 1991,114, 164568.